PROJECT SUMMARY/ABSTRACT Diabetes affects 24 million Americans, 220 million people worldwide, and is the 6th leading cause of death in the US. Diabetic nephropathy (DN) is the most common cause of kidney failure in the US. This chronic and debilitating disease is characterized by progressive albuminuria, declining glomerular filtration rate (GFR), and increased risk for cardiovascular disease, eventually requiring dialysis. Clinical trials demonstrate that the combination therapy of angiotensin converting enzyme (ACE) inhibition and angiotensin (ANG) type 1 receptor blockade provides greater renoprotection than ACE inhibitor alone in human DN suggesting that ACE-independent pathways for ANGII formation are of major significance. Upregulated chymase expression has been identified in human DN and may contribute to ACE-independent formation of ANGII. Our preliminary findings support a predominant role of chymase-dependent ANGII formation on renal microvascular function in the diabetic kidney. We hypothesize that a switch from ACE-dependent to chymase-dependent intrarenal ANGII production in concert with altered ACE2 activity contribute to the decline in function and progression to end-stage disease in type II diabetes. The proposed studies will determine the contribution of intrarenal production of ANGII via ACE-dependent and ACE-independent pathways to the microvascular and glomerular dysfunction mechanisms involved in the progression of diabetic renal disease. Our hypothesis will be tested in the obese diabetic db/db mouse model which exhibits the principal features of human type II DN with 3 Specific Aims: 1) to establish that chymase-dependent mechanisms contribute to the intrarenal production of ANGII and reduction in renal vascular function in diabetes, 2) to demonstrate that early increases in ACE2 activity lead to renal protection via increased production of the vasodilator ANG1-7 and degradation of the vasoconstrictor ANGII, but that a subsequent reduction in ACE2 contributes to complications in later stages of diabetic renal disease, and 3) to demonstrate that chronic inhibition of chymase reduces the intrarenal formation of ANGII, improves renal and cardiovascular function, and ameliorates progression of diabetic renal disease. In vivo and in vitro experimental approaches will quantitate molecular expression, enzymatic activity, and peptide levels of chymase and the major renin-angiotensin system components, histological markers of renal disease, and cardiovascular and renal physiological function in conscious mice. Afferent arteriole (AA) diameter responses to intrarenally produced ANGII will be measured in blood perfused juxtamedullary nephrons. The results from the proposed studies will identify an alternative mechanism for ANGII production in diabetic renal disease which challenges the existing paradigms underlying currently available therapies. If effective, directed pharmacotherapies targeting chymase-dependent ANGII forming pathways will lead to a novel and more effective approach to decrease DN, hypertension, mortality, and significantly reducing morbidity and ultimately mortality in this patient population.